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EP3010519B1 - Adénovirus oncolytique pour l'utilisation dans le traitement du cancer du cerveau - Google Patents

Adénovirus oncolytique pour l'utilisation dans le traitement du cancer du cerveau Download PDF

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EP3010519B1
EP3010519B1 EP14736238.8A EP14736238A EP3010519B1 EP 3010519 B1 EP3010519 B1 EP 3010519B1 EP 14736238 A EP14736238 A EP 14736238A EP 3010519 B1 EP3010519 B1 EP 3010519B1
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tumor
oncolytic adenovirus
glioma
adenovirus
patient
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EP3010519A1 (fr
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Juan Fueyo
Candeleria MANZANO-GOMEZ
Charles Conrad
Fred Lang
W.K. Alfred Yung
Frank TUFARO
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University of Texas System
DNAtrix Inc
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University of Texas System
University of Texas at Austin
DNAtrix Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the invention generally relates to the field of medicine and oncology. More particularly, it concerns compositions and methods of treating gliomas in a patient using oncolytic adenoviruses.
  • tumor suppressor genes as opposed to proto-oncogenes, is to antagonize cellular proliferation.
  • a tumor suppressor gene is inactivated, for example by point mutation or deletion, the cell's regulatory machinery for controlling growth is upset. Mutations and/or loss of function in the retinoblastoma tumor suppressor gene have been associated with tumor formation.
  • brain tumors are metastases to the brain from a primary tumor outside of the central nervous system (CNS). Brain tumors derived from metastases are typically more common than primary tumors of the brain. The most common primary tumors that metastasize to the brain are lung, breast, melanoma, and kidney. These brain metastases are usually in multiple sites, but solitary metastases may also occur.
  • Gene therapy is a promising treatment for brain tumors including gliomas because conventional therapies typically fail and are toxic.
  • identification of genetic abnormalities contributing to malignancies is providing crucial molecular genetic information to aid in the design of gene therapies.
  • Genetic abnormalities indicated in the progression of tumors include the inactivation of tumor suppressor genes and the overexpression of numerous growth factors and oncogenes.
  • Tumor treatment may be accomplished by supplying a polynucleotide encoding a therapeutic polypeptide or other therapeutic that target the mutations and resultant aberrant physiologies of tumors. It is these mutations and aberrant physiology that distinguishes tumor cells from normal cells.
  • a tumor-selective virus would be a promising tool for gene therapy.
  • Targeting the Rb pathway has noted relevance for the treatment of gliomas because abnormalities of the p16/Rb/E2F pathway are present in most gliomas (Fueyo et al ., 1998a; Gomez-Manzano et al ., 1998). Targeting this pathway by replacement of lost tumor suppressor activity through the transfer of p16 and Rb genes has produced cytostatic effects (Fueyo et al ., 1998a; Gomez-Manzano et al ., 1998).
  • Additional treatments include an adenovirus with therapeutic capabilities or with an ability to be tracked in vivo.
  • an oncolytic adenovirus for use in one or more of: (i) a method for achieving greater than 25% reduction in tumor burden when treating a glioma in a human patient without producing of an adverse event resulting from said oncolytic adenovirus that is sufficient to cause termination of said treatment; (ii) a method for achieving a six-month progression-free survival when treating a glioma in a human patient without producing of an adverse event resulting from said oncolytic adenovirus that is sufficient to cause termination of said treatment; and (iii) a method for achieving tumor necrosis when treating a glioma in a human patient without producing of an adverse event resulting from said oncolytic adenovirus that is sufficient to cause termination of said treatment, wherein the method comprises contacting the glioma, in the human patient identified as having the glioma, with the oncolytic adenovirus, and wherein the oncolytic a
  • the subject may also exhibit an autoimmune response against said glioma.
  • the tumor response may include less defined tumor borders as determined by contrast MRI.
  • the oncolytic adenovirus may be a ⁇ 24 adenovirus.
  • a method of identification of a patient having a glioma may comprise tumor imaging, and said method may further comprise obtaining a biopsy of said tumor after identification of a patient having glioma and before treatment with the oncolytic adenovirus.
  • the glioma may be an astrocytoma, an oligodendroglioma, an anaplastic glioma, a glioblastoma, an ependymoma, a meningioma, a pineal region tumor, a choroid plexus tumor, a neuroepithelial tumor, an embryonal tumor, a peripheral neuroblastic tumor, a tumor of cranial nerves, a tumor of the hemopoietic system, a germ cell tumor, or a tumor of the sellar region.
  • the glioma may be recurrent, and/or the glioma may be failed one or more primary glioma therapies.
  • the glioma may be resectable, or not resectable.
  • the glioma may be resected following said treatment.
  • the post-resection tumor bed may be treated with said oncolytic adenovirus.
  • the glioma may be contacted with the adenovirus by delivery of the adenovirus intracranially into the patient.
  • the delivery may comprise intratumoral injection, may comprise multiple injections, such as where a post-resection catheter is implanted into said patient and said oncolytic adenovirus is delivered via said catheter.
  • the oncolytic adenovirus may be administered via slow infusion over a period of minimum 10 minutes with a needle.
  • the oncolytic adenovirus may be administered at stereotactly into more than one site in a glioma in said patient.
  • the dose may be about 10 3 to about 10 15 viral particles, about 10 5 to about 10 12 viral particles are administered to the patient, or about 10 7 to about 10 10 viral particles administered to the patient.
  • the treatment may comprise dosing at 1 ⁇ 10 7 , 3 ⁇ 10 7 , 1 ⁇ 10 8 , 3 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 1 ⁇ 10 10 , and 3 ⁇ 10 10 viral particles, including a dose escalation.
  • the method may further comprise administering to the patient a second therapy, wherein the second therapy is anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresive agents, or gene therapy with a therapeutic polynucleotide.
  • the second therapy may be administered to the patient before administration of the composition comprising the oncolytic adenovirus, administered to the patient at the same time as administration of the composition comprising the oncolytic adenovirus, or administered to the patient after administration of the composition comprising the oncolytic adenovirus.
  • the chemotherapy may comprise an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite.
  • the second therapy may in particular comprise radiotherapy and temozolomide.
  • the subject may be further selected based on the presence of a Th1 response.
  • the Th1 response may be is characterized by an increase in antigen-specific interferon-gamma (IFN- ⁇ ), IL-12, and complement-fixing antibodies.
  • IFN- ⁇ antigen-specific interferon-gamma
  • IL-12 IL-12
  • complement-fixing antibodies complement-fixing antibodies
  • an oncolytic adenovirus for use in one or more of: (i) a method for treating a glioma in a human patient and achieving a clinical benefit in 30% of said patients, with clinical benefit defined by complete responders + partial responders plus stable disease; (ii) a method for treating a glioma in a human patient and achieving a 25% six-month progression-free survival; and (iii) a method for treating a glioma in a human patient and achieving a 12 month median survival for responders, with responders defined by complete responders + partial responders, wherein the method comprises contacting the gliomas of a population of the human patients that have been identified as having glioma with the oncolytic adenovirus, and wherein the oncolytic adenovirus is an oncolytic adenovirus with E1A polypeptide that cannot bind Rb, and comprises a fiber protein with an RGD amino acid inserted in the HI domain.
  • the oncolytic adenovirus may be a ⁇ 24 adenovirus.
  • Step (a) may comprise tumor imaging, and said method may further comprise obtaining a biopsy of said tumore after step (a) and before step (b).
  • the glioma may be an astrocytoma, an oligodendroglioma, an anaplastic glioma, a glioblastoma, an ependymoma, a meningioma, a pineal region tumor, a choroid plexus tumor, a neuroepithelial tumor, an embryonal tumor, a peripheral neuroblastic tumor, a tumor of cranial nerves, a tumor of the hemopoietic system, a germ cell tumor, or a tumor of the sellar region.
  • the glioma may be recurrent, and/or the glioma may be failed one or more primary glioma therapies.
  • the glioma may be resectable, or not resectable.
  • The may be glioma resected following said treatment.
  • the post-resection tumor bed may be treated with said oncolytic adenovirus.
  • the glioma may be contacted with the adenovirus by delivery of the adenovirus intracranially into the patient.
  • the delivery may comprise intratumoral injection, may comprise multiple injections, such as where a post-resection catheter is implanted into said patient and said oncolytic adenovirus is delivered via said catheter.
  • the oncolytic adenovirus may be administered via slow infusion over a period of minimum 10 minutes with a needle.
  • the oncolytic adenovirus may be administered at stereotactly into more than one site in a glioma in said patient.
  • the dose may be about 10 3 to about 10 15 viral particles, about 10 5 to about 10 12 viral particles are administered to the patient, or about 10 7 to about 10 10 viral particles administered to the patient.
  • the method may further comprise administering to the patient a second therapy, wherein the second therapy is anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresive agents, or gene therapy with a therapeutic polynucleotide.
  • the second therapy may be administered to the patient before administration of the composition comprising the oncolytic adenovirus, administered to the patient at the same time as administration of the composition comprising the oncolytic adenovirus, or administered to the patient after administration of the composition comprising the oncolytic adenovirus.
  • the chemotherapy may comprise an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite.
  • the subject may be further selected based on the presence of a Th1 response.
  • the Th1 response may be is characterized by an increase in antigen-specific interferon-gamma (IFN- ⁇ ), IL-12, and complement-fixing antibodies.
  • IFN- ⁇ antigen-specific interferon-gamma
  • IL-12 IL-12
  • complement-fixing antibodies complement-fixing antibodies
  • Embodiments discussed in the context of a methods and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method may be applied to other methods of the invention as well.
  • malignant tumors that are intrinsically resistant to conventional therapies are significant therapeutic challenges.
  • Such malignant tumors include, but are not limited to malignant gliomas, which are the most abundant primary brain tumors having an annual incidence of 6.4 cases per 100,000 (CBTRUS, 2002-2003).
  • CBTRUS cancer-related gliomas
  • These neurologically devastating tumors are the most common subtype of primary brain tumors and are one of the deadliest human cancers.
  • GBM glioblastoma multiforme
  • median survival duration for patients ranges from 9 to 12 months, despite maximum treatment efforts (Hess et al ., 1999).
  • a prototypic disease, malignant glioma is inherently resistant to current treatment regimens (Shapiro and Shapiro, 1998).
  • the ⁇ 24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24-base-pair deletion within the CR2 portion of the E1A gene. Significant antitumor effects of ⁇ 24 have been shown in cell culture systems and in malignant glioma xenograft models.
  • Oncolytic adenoviruses include conditionally replicating adenoviruses (CRADs), such as Delta 24, which have several properties that make them candidates for use as biotherapeutic agents.
  • CRADs conditionally replicating adenoviruses
  • Delta 24 have several properties that make them candidates for use as biotherapeutic agents.
  • One such property is the ability to replicate in a permissive cell or tissue, which amplifies the original input dose of the oncolytic virus and helps the agent spread to adjacent tumor cells providing a direct antitumor effect.
  • adenovirus is a 36 kb, linear, double-stranded DNA virus (Grunhaus and Horwitz, 1992).
  • Adenoviral infection of host cells results in adenoviral DNA being maintained episomally, which reduces the potential genotoxicity associated with integrating vectors.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • a particular form of the ⁇ 24 virus is DNX-2401 (DNATrix, Houston TX) is a conditionally-replicating adenovirus (AdV) vector type 5 for intratumoral administration that contains a 24 bp deletion (bp 923-946; the Rb-binding domain) in the E1A gene and the insertion of an RGD integrin-binding motif (4C peptide: Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys; SEQ ID NO: 1) in the HI loop of the Ad fiber.
  • AdV conditionally-replicating adenovirus
  • AdV adenovirus vector type 5 for intratumoral administration that contains a 24 bp deletion (bp 923-946; the Rb-binding domain) in the E1A gene and the insertion of an RGD integrin-binding motif (4C peptide: Cys-Asp-Cys-Arg-Gly-Asp-
  • DNX-2401 enters cells via both the normal adenovirus receptor (CAR) as well as the RGD-binding integrins normally expressed only on tumor cells and neovasculature. This is a significant improvement upon previous generation adenoviruses that had to rely on the CAR receptor for activity.
  • This RGD-4C peptide (CDCRGDCFC; SEQ ID NO: 1) has been shown to bind with high affinity to the RGD-binding ( ⁇ V ⁇ 3 and ⁇ V ⁇ 5) integrins present on the surface of many cell types, including tumor cells.
  • RGD-binding integrins have been shown to be expressed in tumor vasculature and on glioma cells but not in normal brain, thereby providing a basis for greatly increased and selective infection of glioblastoma by DNX-2401.
  • DNX-2401 replication is restricted to cells with defects in the Rb pathway, the primary control pathway for cellular division. Because virtually all tumor cells, including >90% of glioblastomas, are defective in the Rb/p16 pathway and already in the cell cycle, DNX-2401 replicates in and kills these tumor cells selectively and efficiently. This high degree of selectivity is accomplished by the deletion of the 24 bp Rb binding domain normally present in the virus E1 protein. A major function of this region is to enable adenovirus replication in healthy cells that have normal Rb function. The deletion of this region causes DNX-2401 to be able to replicate only in tumor cells with Rb pathway defects.
  • Rb -deficient tumor cells Most normal human brain cells are usually quiescent. Cells in the central nervous system (CNS) rarely divide, and these cells are specifically triggered to divide in a limited fashion. Tight regulatory controls have evolved which strictly limit cells from undergoing cell division. The p16/Rb/E2F pathway is an important pathway for maintaining the non-dividing status of fully differentiated cell or negatively regulates the cell-cycle progression of dividing normal cells.
  • Human adenovirus normally infects human cells, which are quiescent (nondividing) or dividing cells (normal or cancer cells). Upon introduction of this virus into a human cell (viral infection), the adenovirus DNA is immediately transcribed by the synthesis of E1A adenoviral protein. The CR2 region of E1A protein interacts specifically with Rb protein and leads to release of E2F, forcing cell entry into S-phase (the DNA Synthesis phase) of the cell cycle and maintaining the cell in the dividing cycle. This series of events effectively commandeers the host cell exclusively for the purpose of expressing virally encoded proteins. Active production of adenoviral particles depends on this ability to drive cells into an active mode of replication, a critical feature of oncolytic viruses.
  • tumor cells provide a replicating environment that favors such activity. Mutations in critical sequences of the viral genome render the adenovirus unable to bind to and inactivate tumor suppressor proteins. These modified adenoviruses are able to replicate exclusively in cells lacking a functional target tumor suppressor gene (tumor cells only).
  • an E1A protein with a 24 base pair deletion in the CR2 region prevents the protein from binding to and inactivating Rb.
  • This attenuated E1A-mutant adenovirus is unable to replicate within normal quiescent cells that have a funtionally active Rb pathway.
  • tumor cells are permissive to viral replication, which in turn efficiently invade and lyse human glioma cells both in vitro and in vivo.
  • the oncolytic potential of ⁇ 24 is dramatic compared with other conditionally replication-deficient adenoviruses, such as Onyx-015.
  • the effects of ⁇ 24 in a mouse xenograft intracranial glioma tumor model are shown in FIG 2 .
  • the curve representing RA55 carries the deletion in the E1B region as in Onyx-015.
  • the oncolytic adenovirus does not have the same degree of potency as ⁇ 24 at comparable doses used (in this case 1 x 10 8 pfu).
  • the negative control ⁇ 24 that is inactivated by ultraviolet exposure.
  • ⁇ 24 has been demonstrated in various human tumor cell lines and in animal xenograft models with known defects of the p16/Rb/E2F pathway. Permissive replication of ⁇ 24 in cell lines with p16/Rb/E2F defects is contrasted with the highly attenuated replication in normal astrocytes and normal quiescent fibroblasts. Additionally, the activity of this virus is attenuated when introduced into tumor cells in which Rb has been functionally restored through stable or transient transfection techniques.
  • gliomas do not metastasize, and therefore an efficient local approach should be enough to cure the disease.
  • every glioma harbors several populations of cells expressing different genetic abnormalities (Sidransky et al ., 1992; Collins and James, 1993; Furnari et al ., 1995; Kyritsis et al ., 1996).
  • the spectrum of tumors sensitive to the transfer of a single gene to cancer cells may be limited.
  • replication competent adenoviruses can infect and destroy cancer cells that are arrested in G 0 . Since gliomas invariably include non-cycling cells, this property is important.
  • the pl6-Rb pathway is abnormal in the majority of gliomas (Hamel et al ., 1993; Henson et al ., 1994; Hirvonen et al ., 1994; Jen et al ., 1994; Schmidt et al ., 1994; Costello et al ., 1996; Fueyo et al ., 1996b; Kyritsis et al ., 1996; Ueki et al ., 1996; Costello et al ., 1997), thus making the ⁇ 24 strategy appropriate for most of these tumors.
  • the loss of the retinoblastoma tumor suppressor gene function has been associated with the causes of various types of tumors and is not limited to treatment of gliomas.
  • an E1A mutation (e.g., a ⁇ 24 mutation in E1A) may be used in combination with mutations in the E1B region of the same adenovirus, thus producing a double mutant adenovirus.
  • an adenovirus may comprise a ⁇ 24 mutation and a deletion in the E1B region that prevents expression or function of the E1B55kD protein.
  • the E1B55kD protein has been shown to bind to and inactivate p53.
  • the E1B region mutation may include a deletion of adenovirus sequences from 2426bp to 3328bp of genebank accession number NC_001406, which is incorporated herein by reference.
  • an oncolytic adenovirus may be used as an adenovirus expression vector.
  • Adenovirus expression vector is meant to include those vectors containing adenovirus sequences sufficient to (a) support packaging of the vector and (b) to express a polynucleotide that has been cloned therein. The insertion position of a polynucleotide encoding a heterologous polypeptide of interest within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the polypeptide of interest may be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al ., (1986) or other region that are not essential for viral replication in the target cell.
  • Traditional methods for the generation of adenoviral particles is co-transfection followed by subsequent in vivo recombination of a shuttle plasmid and an adenoviral helper plasmid into either 293 or 911 cells (Introgene, The Netherlands).
  • Modifications of oncolytic adenovirus described herein may be made to improve the ability of the oncolytic adenovirus to treat cancer.
  • the present invention also includes any modification of oncolytic adenovirus that improves the ability of the adenovirus to target neoplastic cells. Included are modifications to oncolytic adenovirus genome in order to enhance the ability of the adenovirus to infect and replicate in cancer cells by altering the receptor binding molecules.
  • Cell surface receptors are attractive candidates for the targeted therapy of cancer.
  • the absence or the presence of low levels of the coxsackievirus and adenovirus receptor (CAR) on several tumor types can limit the efficacy of the oncolytic adenovirus.
  • Various peptide motifs may be added to the fiber knob, for instance an RGD motif (RGD sequences mimic the normal ligands of cell surface integrins), Tat motif, poly-lysine motif, NGR motif, CTT motif, CNGRL motif, CPRECES motif or a strept-tag motif (Rouslahti and Rajotte, 2000).
  • a motif can be inserted into the HI loop of the adenovirus fiber protein. Modifying the capsid allows CAR-independent target cell infection.
  • Peptide sequences that bind specific human glioma receptors such as EGFR or uPR may also be added.
  • Specific receptors found exclusively or preferentially on the surface of cancer cells may used as a target for adenoviral binding and infection, such as EGFRvIII.
  • Rb is a tumor suppressor gene whose loss of function is associated with tumor formation.
  • Retinoblastoma protein or Rb refers to the polypeptide encoded by the retinoblastoma gene (Rb).
  • the retinoblastoma gene is located at 13ql4 in humans and encodes a protein of approximately 110 kiloDaltons (kD).
  • Unphosphorylated Rb inhibits cell proliferation by sequestering transcription factors (e.g., E2F) and arresting cells in G 1 of the cell cycle. Transcription factors are released from Rb when Rb is phosphorylated.
  • E1A Several viral oncoproteins target Rb for inactivation in order to facilitate viral replication. These proteins include adenovirus E1A, SV40 large T antigen, and papillomavirus E7.
  • the E1A protein is one of the first virus-specific polypeptides synthesized after adenoviral infection and is required for viral replication to occur (Dyson and Harlow, 1992; Flint and Shenk, 1997). Interaction of the Rb protein and the E1A protein results in release of E2F from pre-existing cellular E2F-Rb complexes. E2F is then free to activate transcription from E2 promoters of adenovirus and E2F regulated genes of an infected cell. The transcriptional activation of these cellular genes in turn helps to create an environment suitable for viral DNA synthesis in otherwise quiescent cells (Nevins, 1992).
  • E1A Two segments of E1A are important for binding Rb; one includes amino acids 30-60 and the other amino acids 120-127 (Whyte et al ., 1988; Whyte et al ., 1989). Deletion of either region prevents the formation of detectable ElA/Rb complexes in vitro and in vivo (Whyte et al ., 1989).
  • An adenovirus containing a Delta 24 mutation produces an E1A protein that cannot bind Rb, causing an infected cell to remain in G 0 .
  • a mutant Rb pathway and a mutant E1A, along with E2F activation are necessary for ⁇ 24 adenoviral transcription.
  • Retinoblastoma (Rb) pathway refers the interaction of a group of regulatory proteins that interact with Rb or other proteins that interact with Rb in regulating cell proliferation (for review see Kaelin, 1999). Proteins within the Rb pathway include, but are not limited to, Rb, the E2F family of transcription factors, DRTF, RIZ286, MyoD287, cAb1288, MDM2289, hBRG1/hBRM, pl6, p107, pl30, c-Abl tyrosine kinase and proteins with conserved LXCXE motifs, cyclin E-cdk 2, and cyclin D-cdk 4/6.
  • E2F Phosphorylation of Rb releases E2F, which is bound to unphosphorylated Rb.
  • E2F stimulates cyclin E transcription and activity, which results in more Rb phosphorylation.
  • Unphosphorylated Rb acts as a tumor suppressor by binding to regulatory proteins that increase DNA replicaiton, such as E2F (The Genetic Basis of Human Cancer, Vogelstein and Kinzler eds., 1998).
  • Defective retinoblastoma pathway refers to inactivation, mutation, or deletion of the Rb or the inability of the upstream or downstream regulatory proteins that interact with Rb to regulate cell proliferation due to a mutation or modification of one or more proteins, protein activities, or protein-protein interactions. Mutations causing a defective Rb pathway include, but are not limited to inactivating mutations in Rb, INK4 proteins, and CIP/KIP and activating mutations in the cyclin genes, such as cyclin D/cdk 4, 6 and cyclin E, cdk 2.
  • Rb associated tumors include gliomas, sarcomas, tumors of the lung, breast, ovary, cervix, pancreas, stomach, colon, skin, larynx, bladder and prostate.
  • the present invention involves the treatment of brain tumors, including tumor cells with a disrupted Rb pathway. It is contemplated that a wide variety of brain tumors may be treated using the methods and compositions of the invention, including glioblastoma, anaplastic astrocytoma, and gliosarcoma.
  • glioma refers to a tumor originating in the neuroglia of the brain or spinal cord.
  • Gliomas are derived form the glial cell types such as astrocytes and oligodendrocytes, thus gliomas include astrocytomas and oligodendrogliomas, as well as anaplastic gliomas, glioblastomas, and ependymomas.
  • Astrocytomas and ependymomas can occur in all areas of the brain and spinal cord in both children and adults.
  • Oligodendrogliomas typically occur in the cerebral hemispheres of adults. Gliomas account for 75% of brain tumors in pediatrics and 45% of brain tumors in adults.
  • the remaining percentages of brain tumors are meningiomas, ependymomas, pineal region tumors, choroid plexus tumors, neuroepithelial tumors, embryonal tumors, peripheral neuroblastic tumors, tumors of cranial nerves, tumors of the hemopoietic system, germ cell tumors, and tumors of the sellar region.
  • response and progression criteria are defined as those terms were adopted by the World Health Organization and adapted for brain tumors, using Macdonald criteria (Macdonald et al ., 1990), and are determined using bi-dimensional measurements of contrast-enhancing lesions (reduction on longest cross diameter of a lesion on an MRI scan):
  • Glioblastoma is a devastating primary high grade malignant glioma resistant to conventional therapies.
  • Current intervention such as surgery, radiotherapy and chemotherapy, extends overall median survival to approximately 14.6 months.
  • Many new compounds even when tested in combination, have failed to improve overall survival or lead to a useful clinical response.
  • High-grade malignant gliomas are highly vascular and infiltrative tumors, and are therefore inclined to recur despite surgical resection. Treatment options are limited for newly-diagnosed as well as recurrent disease, and are especially limited for patients who have tumors that are not surgically accessible. Furthermore, while 80% or more of glioblastoma recurrences occur in the same area as the original tumor, additional radiation therapy is often precluded because of toxicity concerns. Temozolomide is approved for treating newly-diagnosed glioblastoma and recurrent anaplastic astrocytoma and bevaciumab was more recently approved for treating recurrent glioblastoma. These drugs are systemically delivered, and must be administered as a multi-dose regimen. Temozolomide is used most effectively as an adjuvant to surgery or radiotherapy. By contrast, bevacizumab is often administered on its own for recurrent disease, or experimentally, in combination with existing chemotherapies such as irinotecan.
  • the therapeutic regimens may include a combination of these therapies, often involve the concurrent prescription of non - anti-angiogenic treatments, and can be administered to both patients with newly diagnosed and recurrent disease.
  • Initial results suggests that while several of these agents can modestly prolong 6-month PFS, the potential long-term benefits and impact on survival remain to be demonstrated.
  • Glioblastoma multiforme is the most common malignant primary brain tumor of adults. More than half of these tumors have abnormalities in genes involved in cell cycle control. Often there is a deletion in the CDKN2A or a loss of expression of the retinoblastoma gene.
  • Other types of brain tumors include astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, meningiomas and schwannomas.
  • therapeutic benefit refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his/her condition, which includes treatment of pre-cancer, cancer, and hyperproliferative diseases.
  • a list of nonexhaustive examples of this includes extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition.
  • adenoviral delivery to in vivo and ex vivo situations.
  • viral vectors one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 or 1 x 10 13 infectious particles to the patient in a pharmaceutically acceptable composition as discussed below.
  • Various routes are contemplated for various tumor types. Where discrete tumor mass, or solid tumor, may be identified, a variety of direct, local and regional approaches may be taken.
  • the tumor may be directly injected with the adenovirus.
  • a tumor bed may be treated prior to, during or after resection and/or other treatment(s).
  • Following resection or other treatment(s) one generally will deliver the adenovirus by a catheter having access to the tumor or the residual tumor site following surgery.
  • One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery.
  • a more distal blood supply route also may be utilized.
  • the method of treating cancer includes treatment of a tumor as well as treatment of the region near or around the tumor.
  • residual tumor site indicates an area that is adjacent to a tumor. This area may include body cavities in which the tumor lies, as well as cells and tissue that are next to the tumor.
  • compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention may include classic pharmaceutical preparations. One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells.
  • Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions according to the present invention will be via an appropriate route, but are particularly drawn to intracranial/intratumoral administration. Administration may be by injection or infusion, see Kruse et al. (1994), specifically incorporated by reference, for methods of performing intracranial administration. Such compositions would normally be administered as pharmaceutically acceptable compositions.
  • an effective amount of the therapeutic agent is determined based on the intended goal, for example, elimination of tumor cells.
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • the engineered viruses of the present invention may be administered directly into animals, or alternatively, administered to cells that are subsequently administered to animals.
  • in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture.
  • ex vivo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a living animal.
  • in vivo administration includes all manipulations performed on cells within an animal.
  • the compositions may be administered either in vitro, ex vivo, or in vivo.
  • An example of in vivo administration includes direct injection of tumors with the instant compositions by intracranial administration to selectively kill tumor cells.
  • Intratumoral injection or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors including tumor exposed during surgery.
  • the injection volume will be 1 to 3 cc, preferably 3 cc.
  • the injection volume will be 4 to 10 cc, preferably 5 cc.
  • Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes, preferable 0.2 ml.
  • the viral particles may advantageously be contacted by administering multiple injections to the tumor, spaced at approximately 1 cm intervals.
  • the present invention may be used preoperatively, to render an inoperable tumor subject to resection.
  • the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease.
  • a resected tumor bed may be injected or perfused with a formulation comprising the adenovirus.
  • the perfusion may be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment also is envisioned.
  • Continuous administration preferably via catheterization, also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease.
  • Such continuous perfusion may take place for a period from about 1-2 hr, to about 2-6 hr, to about 6-12 hr, to about 12-24 hr, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment.
  • the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.
  • Treatment regimens may vary as well, and often depend on tumor type, tumor location, disease progression, and health and age of the patient. Obviously, certain types of tumor will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride or Ringer's dextrose.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases.
  • the pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
  • the route is topical, the form may be a cream, ointment, or salve.
  • Tumor cell resistance to various therapies represents a major problem in clinical oncology.
  • One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy, as well as other conventional cancer therapies.
  • One way is by combining such traditional therapies with oncolytic adenovirus therapy.
  • Traditional therapy to treat cancers may include removal of all or part of the affected organ, external beam irradiation, xenon arc and argon laser photocoagulation, cryotherapy, immunotherapy and chemotherapy.
  • the choice of treatment is dependent on multiple factors, such as, 1) multifocal or unifocal disease, 2) site and size of the tumor, 3) metastasis of the disease, 4) age of the patient or 5) histopathologic findings (The Genetic Basis of Human Cancer, 1998).
  • adenoviral therapy could be used in conjunction with anti-cancer agents, including chemo- or radiotherapeutic intervention, as well as radiodiagnositc techniques. It also may prove effective to combine oncolytic virus therapy with immunotherapy.
  • a "target” cell contacting a mutant oncolytic virus and optionally at least one other agent may kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce a hyperproliferative phenotype of target cells.
  • These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the target cell.
  • This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same or different times. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, wherein one composition includes the oncolytic adenvirus and the other includes the second agent.
  • Oncolytic adenoviral therapy may also be combined with immunosuppression.
  • the immunosuppression may be performed as described in WO 96/12406 ).
  • immunosuppressive agents include cyclosporine, FK506, cyclophosphamide, and methotrexate.
  • an oncolytic adenovirus treatment may precede or follow the second agent or treatment by intervals ranging from minutes to weeks.
  • the second agent and oncolytic adenovirus are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the second agent and oncolytic adenovirus would still be able to exert an advantageously combined effect on the cell.
  • oncolytic adenovirus is "A” and the other agent is "B”, as exemplified below:
  • both agents are delivered to a cell in a combined amount effective to kill the cell.
  • Agents or factors suitable for use in a combined therapy are any anti-angiogenic agent and/or any chemical compound or treatment method with anticancer activity; therefore, the term "anticancer agent” that is used throughout this application refers to an agent with anticancer activity.
  • These compounds or methods include alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, DNA antimetabolites, antimitotic agents, as well as DNA damaging agents, which induce DNA damage when applied to a cell.
  • Examples of chemotherapy drugs and pro-drugs include, CPT11, temozolomide, platin compounds and pro-drugs such as 5-FC.
  • Examples of alkylating agents include, inter alia, chloroambucil, cis-platinum, cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone.
  • Topoisomerase I inhibitors encompass compounds such as camptothecin and camptothecin derivatives, as well as morpholinodoxorubicin. Doxorubicin, pyrazoloacridine, mitoxantrone, and rubidazone are illustrations of topoisomerase II inhibitors.
  • RNA/DNA antimetabolites include L-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate, and pyrazofurin; while the DNA antimetabolite group encompasses, for example, ara-C, guanozole, hydroxyurea, thiopurine.
  • Typical antimitotic agents are colchicine, rhizoxin, taxol, and vinblastine sulfate.
  • Other agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • Chemotherapeutic agents function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein.
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, bleomycin, 5-fluorouracil (5-FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), podophyllotoxin, verapamil, and even hydrogen peroxide.
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • the cells of a precancerous lesion or tumor cells with an agent in addition to the oncolytic adenovirus. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves.
  • the cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as adriamycin, bleomycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, podophyllotoxin, verapamil, or more preferably, cisplatin.
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with an oncolytic adenovirus.
  • Cisplatinum agents such as cisplatin, and other DNA alkylating agents may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 for 5 days every three weeks for a total of three courses.
  • Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Bleomycin and mitomycin C are other anticancer agents that are administered by injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • a typical dose of bleomycin is 10 mg/m 2 , while such a dose for mitomycin C is 20 mg/m 2 .
  • Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally.
  • nucleic acid precursors and subunits also lead to DNA damage.
  • nucleic acid precursors have been developed.
  • agents that have undergone extensive testing and are readily available are agents that have undergone extensive testing and are readily available.
  • agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
  • 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used or as alternative 5-FC may be administered and converted in a target tissue or target cell.
  • ⁇ -rays X-rays
  • X-rays X-rays
  • UV-irradiation UV-irradiation
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • Immunotherapy may be used as part of a combined therapy, in conjunction with mutant oncolytic virus-mediated therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
  • Antibodies specific for CAR, integrin or other cell surface molecules may be used to identify cells that the adenovirus could infect well.
  • CAR is an adenovirus receptor protein. The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalization.
  • oncolytic adenovirus therapies with chemo- and radiotherapies
  • combination with other gene therapies will be advantageous.
  • targeting of an oncolytic adenovirus in combination with the targeting of p53 at the same time may produce an improved anti-cancer treatment.
  • Any tumor-related gene or nucleic acid encoding a polypeptide conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
  • Anti-angiogenic therapies may also be combined advantageously with the oncolytic adenovirus therapies disclosed herein.
  • Bevacizumab (Avastin®), Genentech/Roche) is an angiogenesis inhibitor, a drug that slows the growth of new blood vessels. It is licensed to treat various cancers, including colorectal, lung, breast (outside the USA), glioblastoma (USA and Japan), kidney and ovarian.
  • Bevacizumab is a humanized monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A).
  • VEGF-A is a chemical signal that stimulates angiogenesis in a variety of diseases, especially in cancer. Bevacizumab was the first clinically available angiogenesis inhibitor in the United States.
  • the therapies described above may be implemented in combination with all types of surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. These types of surgery may be used in conjunction with other therapies, such as oncolytic adenovirus therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, systemic administration, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • the time between such treatment types may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours apart; about 1, 2, 3, 4, 5, 6, or 7 days apart; about 1, 2, 3, 4, or 5 weeks apart; and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart, or more.
  • Rb pathway is required to be defective in the sense that it is not able to repress the transcription-activating activity of E2F.
  • E2F activates the transcription of cellular genes and adenoviral DNA if its activity is not repressed. Examples of ways in which E2F could escape repression include, but are not limited to, Rb not being able to bind E2F ( i.e., E1A binding to Rb), overexpression of E2F, less Rb than E2F and situations in which Rb remains phosphorylated.
  • Th1 polarized immune response in subjects is predictive of successful treatment with the oncolytic adenoviruses of the present invention.
  • the presence of Th2 response may be an indicator of non-response.
  • a particular Th1 marker is IL-12p70.
  • high levels of antibodies again tumor associated antigens such as NLRP4 maybe assessed and if found predicts response to the oncolytic viral therapy.
  • Th1 markers include IL-1 ⁇ , IL-2, IL-8, IL-12, IL-18, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , GMCSF, cleaved caspase 3, neopterin and ⁇ 2-microglobuin.
  • Th1 surface markers include CXCR3, CCR5, CCR1 and IL-12 recepter ⁇ 1 and ⁇ chains.
  • Th2 markers include IL-4, IL-5, IL-6, IL-10, IL-13, TGF ⁇ and phosphorylated STAT3.
  • Th2 surface markers include CXCR4, CCR3, CCR4, CCR7, CCR8, IL-1 receptor and CD30.
  • Tumor associated antigens include BRAF, CABYR, CRISP3, CSAG3, CTAG2, DHFR, FTHL17, GAGE1, LDHC, MAGEA1, MAGEA3, MAGEA4, MAGEB6, MAPK1, MICA, MUC1, NLPR4, NYES01, P53, PBK, PRAME, SOX2, SPANXA1, SSX2, SSX4, SSX5, TSGA10, TSSK6, TULP2, XAGE2 and ZNF165.
  • 1, 2, 3, 4, 5, 6, 7 or all 8 of CABYR, MAGEA1, MAGEA3, MAGEB6, NLPR4, NYES01, PBK, and ZNF165 are examined.
  • Antibodies can be used to detect adenoviral proteins (e.g ., E1A), Rb, and other proteins of the Rb pathway, Th1 response, Th2 response or tumor associated antigens.
  • adenoviral proteins e.g ., E1A
  • Rb e.g., Rb
  • other proteins of the Rb pathway e.g., Th1 response, Th2 response or tumor associated antigens.
  • one or more antibodies may be produced that are immunoreactive with multiple antigens. These antibodies may be used in various diagnostic or therapeutic applications, described herein below.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • Means for preparing and characterizing antibodies are also well known in the art (see, e.g., Harlow and Lane (1988).
  • Certain embodiments of the invention provide antibodies to antigens and translated proteins, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate.
  • a reporter molecule is defined as any moiety which may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.
  • Rb expression or adenoviral gene expression in a population of cells can be determined by western blot analysis using antibodies as probes to adenoviral proteins. The level of viral proteins detected would indicate whether viral protein expression is occurring in the cell.
  • Immunodetection methods for detecting biological components such as protein(s), polypeptide(s) or peptide(s) involved in adenoviral replication or the cellular Rb or p53 pathways may be employed.
  • Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay immunoradiometric assay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot to mention a few.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zee
  • the biological sample analyzed may be any sample that is suspected of containing an antigen, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood and/or serum, although tissue samples or extracts are preferred.
  • an antigen such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood and/or serum, although tissue samples or extracts are preferred.
  • a tumor may be biopsied and the above tests performed upon it to determine the presence or absence of glioma cells, either prior to, during or aftern treatment.
  • An example of a biopsy protocol is as follows. The stereotactic biopsy is the precise introduction of a metal probe into the brain tumor, cutting a small piece of the brain tumor, and removing it so that it can be examined under the microscope. The patient is transported to the MRI or CAT scan suite, and the frame is attached to the scalp under local anesthesia. The "pins" of the frame attach to the outer table of the skull for rigid fixation (frame will not and can not move from that point forward until completion of the biopsy). The scan (MRI or CT) is obtained. The neurosurgeon examines the scan and determines the safest trajectory or path to the target.
  • the spatial co-ordinates of the target are determined, and the optimal path is elected.
  • the biopsy is carried out under general anesthesia. A small incision is created over the entry point, and a small hole is drilled through the skull. The "dura" is perforated, and the biopsy probe is introduced slowly to the target. The biopsy specimen is withdrawn and placed in preservative fluid for examination under the microscope. Often the pathologist is present in the biopsy suite so that a rapid determination of the success of the biopsy can be made.
  • a Phase 1, dose-escalating, two-part study of DNX-2401 for high-grade glioma was initiated under an investigator-sponsored IND at MD Anderson Cancer Center in Houston Texas. To be eligible for the study, patients were required to have histologically-proven, recurrent high-grade malignant glioma.
  • Group A of the study evaluated the direct intratumoral injection of a single dose of DNX-2401 into a growing area of biopsy-confirmed recurrent glioma, while Group B evaluated the injection of a divided dose of virus into the resection bed following glioma excision.
  • the starting dose for both study groups was 10 7 ( e.g., 1 x 10 7 ) viral particles (vp), with a plan to dose escalate in half-log increments up to 3 10 vp.
  • the primary objectives of the study were to determine the safety, tolerability, feasibility, and biological effect of injecting DNX-2401 into human brain tumors in situ.
  • Patients in Group A received direct intratumoral injection through a needle and underwent standard dose escalation by cohort. Tumors may or may not have been surgically resectable.
  • the assigned dose levels were: 1 ⁇ 10 7 , 3 ⁇ 10 7 , 1 ⁇ 10 8 , 3 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 1 ⁇ 10 10 , and 3 ⁇ 10 10 viral particles (vp).
  • Patients were observed for 28 days following virus injection before patients in the next cohort were enrolled and treated.
  • Group B only included patients with resectable tumors. Patients in Group B received direct intratumoral injection through a permanently implanted catheter in the center of the tumor and then underwent standard dose escalation by cohort (i.e., dose levels 1 ⁇ 10 7 , 3 ⁇ 10 7 , 1 ⁇ 10 8 , 3 ⁇ 10 8 , 1 ⁇ 10 9 , 3 ⁇ 10 9 , 1 ⁇ 10 10 , and 3 ⁇ 10 10 viral particles (vp)). Dose escalation for group B was similar to group A, except that group B lagged behind group A by one dose level. Patients in Group A were not included in Group B.
  • DNX-2401 was injected into the residual tumor surrounding the resection cavity (i.e ., intramural injection into the tumor bed).
  • Group A completed the enrollment of 25 subjects in September 2012. The maximum dose achieved was 3 ⁇ 10 10 vp as planned. Enrollment into group B that evaluated DNX-2401 as an adjunct to surgery was initiated later and enrolled 12 subjects with the maximum dose of 3 ⁇ 10 8 vp.
  • Follow-up was scheduled to occur at monthly intervals for 4 months, every 2 months for 2 years, and every 4 months for life thereafter for both treatment groups. Patients were and will be monitored for toxicity and symptoms, and evaluated using magnetic resonance imaging (MRI), spinal tap, and other tests as appropriate based on clinical standards of care for the duration of the study.
  • MRI magnetic resonance imaging
  • Study assessments for both treatment groups were performed at regular time intervals as outlined in the schedule of assessments. Data were recorded in electronic case report forms per MD Anderson standards and intra-institutionally monitored through MD Anderson's IND office approximately every 4 weeks. The data presented are unaudited and should be considered preliminary at this time.
  • Group B (12 enrolled). Patients with histopathologically-confirmed recurrent high-grade glioma were heavily pre-treated by the time of enrollment. All patients had received radiotherapy with concomitant temozolomide.
  • Tumor response to therapy appears to be accompanied by signature changes on contrast MRI. These include early, global changes in contrast pattern ("bunch of grapes") followed in some instances by the emerge of a "thread” pattern or what resembles "soap bubbles.”
  • bunch of grapes early, global changes in contrast pattern
  • thread pattern or what resembles "soap bubbles.”
  • DNX-2401 Treatment of several tumors appear to progress and have less defined borders. This is now thought to be caused by inflammation, such as that seen with other immunotherapy products. This will then change to the more distinct, smaller tumor, which in some instances goes on to a complete response.
  • Anti-tumor response to treatment is an especially important endpoint in this disease, as displacement of normal brain tissue due to rapidly growing tumor eventually results in severe disability and death.
  • DNX-2401 has the potential to be safer and more effective than current therapies for recurrent gliobastoma.
  • a response to DNX-2401 therapy may be based on a cellular versus humoral immune response. Because of this, the inventors looked at the cytokine profile of responders versus non-responders with the expectation that strong responders would exhibit more of a Th1 (cytotoxic T8 cell) polarization and non-responders would show a profile more consistent with a Th2 (antibody-producing) profile.
  • Th1 cytotoxic T8 cell
  • Th2 antibody-producing
  • the Th1 response is characterized by an increase in antigen-specific interferon-gamma (IFN- ⁇ ), IL-12, and complement-fixing antibodies
  • the Th2 phenotype is characterized by production of IL-4, IL-5, IL-10, and an increase in IgE, IgA, and overall IgG antibodies. This expectation was confirmed by cytokine expression (using ELISA semiquantitative MSD assays).
  • antibodies in patient sera has several advantages when compared to other more conventional biomarker classes.
  • serum is a readily accessible tissue requiring relatively non-invasive sampling.
  • antibodies provide an amplified response and their relative abundance enables early warning or detection of small changes.
  • a tissue biopsy is not required, which is both invasive, unpleasant to the patient and depending on the tumor accessibility, often contains a mixture of various cell types.
  • Patient #12 (56 year-old white female) was diagnosed that led to tumor resection and subsequent treatment with chemotherapy that consisted of temozolomide and dasatinib, and radiotherapy. Sshe was enrolled in Group A (intratumoral administration of DNX-2401) and randomized to Cohort 3. She received 4 intratumoral injections/1 mL of DNX-2401 at a total dose of 1 ⁇ 10 8 viral particles (vp). Clinically, the patient has done very well. All neurologic symptoms subsequently resolved over the first 6 months. She did have a slight increase in the overall size of the brain lesion that had the appearance of pseudo-progression/inflammation; however, this was followed by continuous tumor shrinkage.
  • Patient #33 Patient #33 (40 year-old white female) underwent primary tumor resection identifying gliobastoma. She received temozolomide chemotherapy and radiotherapy. She was enrolled in Group A (intratumoral administration of DNX-2401) and randomized to Cohort 7. She received 4 intratumoral injections/mL of DNX-2401 at a total dose of 1 ⁇ 10 10 vp. She did well during the month immediately following injection, with neurological symptoms resolving especially expressive aphasia and partial complex seizures. Additionally, the contrast-enhancing mass as well as the FLAIR abnormality on serial MR scans had virtually disappeared. The patient has not experienced a serious adverse event during the study. Overall, the patient appears to have had a complete response by McDonald criteria. Since DNX-2401 administration, the patient is currently alive and doing well. To date, she is neurologically symptom-free 16 months post injection of DNX-2401.
  • Patient #42 Patient #42 (white female) underwent primary tumor resection that confirmed glioblastoma. She received radiotherapy and also received 4 courses of chemotherapy as follows: temozolomide, memantine temozolomide again, and macitentan. She was enrolled in Group B (intratumoral/intramural administration of DNX-2401) and randomized to Cohort 4. She received one intratumoral injection/mL of DNX-2401 at a total dose of 3 ⁇ 10 8 vp, followed by 10 intramural injections/1 mL of DNX-2401 at a total dose of 3 ⁇ 10 8 vp. Following resection, there was no measurable disease. During study participation she has not experienced a serious adverse event. Patient is currently alive and doing well.

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Claims (15)

  1. Adénovirus oncolytique destiné à être utilisé dans l'un ou plusieurs :
    (i) d'un procédé permettant d'obtenir une réduction de plus de 25 % de la charge tumorale lors du traitement d'un gliome chez un patient humain sans entraîner d'effet indésirable résultant dudit adénovirus oncolytique, lequel effet indésirable est suffisant pour provoquer l'arrêt dudit traitement ;
    (ii) d'un procédé permettant d'obtenir une survie sans progression de la maladie à six mois lors du traitement d'un gliome chez un patient humain sans entraîner d'effet indésirable résultant dudit adénovirus oncolytique, lequel effet indésirable est suffisant pour provoquer l'arrêt dudit traitement ; et
    (iii) d'un procédé permettant d'obtenir une nécrose tumorale lors du traitement d'un gliome chez un patient humain sans entraîner d'effet indésirable résultant dudit adénovirus oncolytique, lequel effet indésirable est suffisant pour provoquer l'arrêt dudit traitement,
    dans lequel le procédé comprend la mise en contact du gliome, chez le patient humain identifié comme étant atteint du gliome, avec l'adénovirus oncolytique, et dans lequel l'adénovirus oncolytique est un adénovirus oncolytique comportant un polypeptide E1A qui ne peut pas se lier à Rb, et comprenant une protéine fibreuse comportant un acide aminé RGD inséré dans le domaine H1.
  2. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, l'adénovirus oncolytique étant un adénovirus Δ24.
  3. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel le patient est identifié comme étant atteint d'un gliome par un procédé qui comprend l'imagerie tumorale, et ledit procédé comprend en outre la réalisation d'une biopsie de ladite tumeur après l'imagerie et avant le traitement avec l'adénovirus oncolytique.
  4. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel ledit gliome est :
    (a) résécable, le procédé comprenant de préférence la résection du gliome après le traitement avec l'adénovirus oncolytique, et un lit tumoral post-résection étant de manière davantage préférée traité avec ledit adénovirus oncolytique, et un cathéter post-résection étant de manière davantage préférée encore implanté dans ledit patient et ledit adénovirus oncolytique étant administré par l'intermédiaire dudit cathéter ;
    (b) non résécable, et le procédé comprenant de préférence la résection du gliome après le traitement avec l'adénovirus oncolytique.
  5. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel le gliome est mis en contact avec l'adénovirus par administration de l'adénovirus au patient par voie intracrânienne, l'administration comprenant par exemple une injection intratumorale et de multiples injections intratumorales étant éventuellement effectuées.
  6. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel le gliome est un astrocytome, un oligodendrogliome, un gliome anaplasique, un glioblastome, un épendymome, un méningiome, une tumeur de la région pinéale, une tumeur du plexus choroïde, une tumeur neuroépithéliale, une tumeur embryonnaire, une tumeur neuroblastique périphérique, une tumeur des nerfs crâniens, une tumeur du système hémopoïétique, des tumeurs des cellules germinales ou une tumeur de la région sellaire, et dans lequel le gliome est de préférence un glioblastome.
  7. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, l'adénovirus oncolytique étant administré :
    (a) par perfusion lente au moyen d'une aiguille pendant au moins 10 minutes ; ou
    (b) stéréotactiquement dans plus d'un site dans un gliome chez ledit patient.
  8. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel le procédé comprend en outre l'administration au patient d'une seconde thérapie, dans lequel la seconde thérapie est une thérapie anti-angiogénique, une chimiothérapie, une immunothérapie, une chirurgie, une radiothérapie, des agents immunosuppresseurs ou une thérapie génique avec un polynucléotide thérapeutique, et éventuellement :
    (a) dans lequel la seconde thérapie est administrée au patient avant l'administration de la composition comprenant l'adénovirus oncolytique ;
    (b) dans lequel la seconde thérapie est administrée au patient en même temps que l'administration de la composition comprenant l'adénovirus oncolytique ;
    (c) dans lequel la seconde thérapie est administrée au patient après l'administration de la composition comprenant l'adénovirus oncolytique ; ou
    (d) dans lequel la chimiothérapie comprend un agent alkylant, un inhibiteur mitotique, un antibiotique ou un antimétabolite.
  9. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel environ 103 à environ 1015 particules virales sont administrées au patient, éventuellement environ 105 à environ 1012 particules virales ou environ 107 à environ 1010 particules virales.
  10. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel ledit sujet est en outre sélectionné sur la base de la présence d'une réponse Th1, et éventuellement dans lequel ladite réponse Th1 est caractérisée par une augmentation de l'interféron-gamma spécifique de l'antigène (IFN-γ), de l'IL-12 et des anticorps fixant le complément.
  11. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, à être utilisé dans 2 ou plus des procédés i) à iii).
  12. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, à être utilisé dans les 3 procédés i) à iii).
  13. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel ledit gliome est récurrent.
  14. Adénovirus oncolytique destiné à être utilisé dans un procédé selon la revendication 1, dans lequel ledit gliome n'a pas été traité par une ou plusieurs thérapies primaires de gliome.
  15. Adénovirus oncolytique destiné à être utilisé dans l'un ou plusieurs :
    (i) d'un procédé permettant de traiter un gliome dans une population de patients humains, et d'obtenir un avantage clinique chez 30 % desdits patients, un avantage clinique étant défini par des patients présentant une réponse complète + des patients présentant une réponse partielle plus une maladie stable ;
    (ii) d'un procédé permettant de traiter un gliome dans une population de patients humains, et d'obtenir une survie sans progression de la maladie à six mois de 25 % ; et
    (iii) d'un procédé permettant de traiter un gliome dans une population de patients humains, et d'obtenir une survie moyenne à 12 mois pour les patients présentant une réponse, les patients présentant une réponse étant définis par des patients présentant une réponse complète + des patients présentant une réponse partielle,
    le procédé comprenant la mise en contact des gliomes d'une population de patients humains qui ont été identifiés comme étant atteints d'un gliome avec l'adénovirus oncolytique, et l'adénovirus oncolytique étant un adénovirus oncolytique comportant un polypeptide E1A qui ne peut pas se lier à Rb, et comprenant une protéine fibreuse comprenant un acide aminé RGD inséré dans le domaine H1.
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